Internazionali - CNReprints.bice.rm.cnr.it/11441/1/article.pdf · Annali di Stomatologia 2014; V (2): 69-76 69 Biocompatibility of a new pulp capping cement Claudio Poggio, MD, DDS1,

Annali di Stomatologia 2014; V (2): 69-76 69

Biocompatibility of a new pulp capping cement

Claudio Poggio, MD, DDS1,

Matteo Ceci, DMD, PhD1,

Riccardo Beltrami, DMD, PhD2,

Alberto Dagna, DMD, PhD,

Marco Colombo, DMD, PhD1,

Marco Chiesa, DMD, PhD1

1 Department of Clinical, Surgical, Diagnosticand Pediatric Sciences, Section of Dentistry,University of Pavia, Italy

2 Department of Brain and Behavioural Sciences,University of Pavia, Italy

Corresponding author:

Claudio PoggioDepartment of Clinical, Surgical, Diagnosticand Pediatric SciencesSection of Dentistry, Policlinico “San Matteo”,University of Pavia Piazzale Golgi 3,27100 Pavia, ItalyPhone: +39 0382 516257, +39 3398124925Fax: +39 0382 516224 E-mail: [email protected]

Summary

Aim. The aim of the present study was to evaluate

the biocompatibility of a new pulp capping materi-

al (Biodentine, Septodont) compared with refer-

ence pulp capping materials: Dycal (Dentsply),

ProRoot MTA (Dentsply) and MTA-Angelus (An-

gelus) by using murine odontoblast cell line and

Alamar blue and MTT cytotoxicity tests.

Methods. The citocompatibility of murine odonto-

blasts cells (MDPC-23) were evaluated at different

times using a 24 Transwell culture plate by Ala-

mar blue test and MTT assay.

Results. The results were significantly different

among the pulp capping materials tested. Bio-

compatibility was significant different among ma-

terials with different composition.

Conclusions. Biodentine and MTA-based prod-

ucts show lower cytotoxicity varying from calci-

um hydroxide-based material which present high-

er citotoxicity.

Key words: biocompatibility, MTT test, murine

odontoblast, pulp capping materials.

Introduction

Direct pulp capping involves the application of a den-tal material to seal communications between the ex-posed pulp and the oral cavity (mechanical and cari-ous pulp exposures) in an attempt to act as a barrier,protect the dental pulp complex and preserve its vital-ity (1). Inducing hard tissue formation by pulp cells asan ultimate goal of capping material use is widely ac-cepted (2).Several materials such as calcium hydroxide-basedmaterials and more recently mineral trioxide aggre-gate (MTA) are commonly used for this purpose (3,4).Calcium hydroxide is the most popular agent for di-rect and indirect pulp capping and maintaining pulpvitality, given its ability to release hydroxyl (OH) andcalcium (Ca) ions upon dissolution (5,6). Both clini-cally and histologically it has been found to producesatisfactory results in indirect and direct pulp capping,because it is capable of stimulating the formation oftertiary dentin by the pulp. This is documented by ba-sic research and clinical studies with reported suc-cess rates in excess of 80% for direct pulp capping(7,8). Currently, calcium hydroxide products are thebest documented and most reliable materials for di-rect pulp capping and serve as the “gold standard”against which new materials have to be tested (9).Nevertheless, calcium hydroxide has some draw-backs. Poor bonding to dentin, material reabsorption,high solubility and mechanical instability are amongthem. In addition, the formation of reparative dentinemay not be due to the bioinductive capacity of thematerial but due to a defense mechanism by the pulpinduced by the irritant nature of calcium hydroxide(10, 11); the high pH (12.5) of calcium hydroxide sus-pensions causes liquefaction necrosis at the surfaceof the pulp tissue with the formation of a necrotic lay-er at the material-pulp interface (7).Dycal (Dentsply) is a self-setting radiopaque calciumhydroxide-based material used both as a pulp cap-ping agent and as a liner under restorations, cementsand other base materials. Its toxicity to pulp cells iswell documented (12, 13). Portland cements, commonly named mineral trioxideaggregate (MTA) cements (such as ProRoot MTA,MTA-Angelus, Tech Biosealer and others), are thera-peutic, endodontic repair calcium silicate materials in-troduced at first as a grey cement (14). These materi-als promote the proliferation/differentiation of humandental pulp cells (15-17) and show calcified tissue-conductive activity with the ability to encourage newhard tissue formation in terms of dentine bridge devel-

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opment over the exposed pulp (18,19). Compared tocalcium hydroxide materials, MTA has an enhancedinteraction with dental pulp tissue (15) with less pulpinflammation and limited pulp tissue necrosis (18, 20).Several new calcium silicate-based materials have re-cently been developed (21-23), aiming to improvesome MTA drawbacks such as its difficult handlingproperty (24) and long setting time (14). Biodentine(Septodont) is among these materials and it is claimedto be used as a dentine restorative material in additionto endodontic indications similar to those of MTA. Thisagent is characterized by the release of calcium hy-droxide in solution (25, 26), which when in contactwith tissue fluids forms hydroxyapatite (27-29).As pulp capping materials will be in direct contactwith pulp tissue for long periods of time, their biocom-patibility is of particular importance. Several methodsfor the determination of biocompatibility of dental ma-terials have been recommended, but the analysis ofin vitro cellular reactions are generally considered tobe the initial approach (30). This allows for the basicbiological characterization of a material and for analy-sis of the underlying cellular mechanisms.The aim of the present study is to evaluate the bio-compatibility of a new pulp capping material (Bioden-tine, Septodont) compared with reference pulp cap-ping materials: Dycal (Dentsply), ProRoot MTA(Dentsply) and MTA-Angelus (Angelus) by usingmurine odontoblast cell line and Alamar blue andMTT test.

Materials and methods

The following materials were used: Dycal, ProRootMTA, MTA-Angelus and Biodentine. The componentsof each material and its manufacturer are reported inTable 1.Dycal, a two-paste system made of a base paste anda catalyst paste (13), was prepared following themanufacturer’s instructions by mixing equal amounts

of catalyst paste and base paste. ProRoot MTA andMTA-Angelus, composed of white Portland cementand bismuth oxide (31, 32), were prepared followingthe manufacturer’s instructions. Biodentine consistsof a powder in a capsule and liquid in a pipette. Thepowder was mixed with the liquid in a capsule in thetriturator for 30 seconds. Once mixed, Biodentinesets in about 12 minutes. During the setting of the ce-ment calcium hydroxide was formed.

Odontoblast cell line culture condition

The mouse odontoblast cell line (MDPC-23) waskindly provided from Dr Jacques Eduardo (Dept. Cari-ology, Restorative Sciences, Endodontics; Universityof Michigan School of Dentistry). Odontoblast-like cellline (MDPC-23) is recommended for application to invitro studies concerning the biocompatibility of dentalmaterials. Mac Dougall (33), reported that the immor-talized mouse odontoblast cell line is positioned inthe periphery of the pulp and are the first cells affect-ed by dental materials. MDPC-23 cells were cultured in DMEM medium(Biowhittaker, Italy) supplemented with 10% fetalbovine serum (FBS), 2% glutamine, 2% sodium pyru-vate, 1% amphotericin and 2% (w/v) streptomycin/penicillin at 37 °C in 5% CO2 atmosphere (34). Thecells were routinely detached using a trypsin-EDTAsolution for 2 minutes at 37°C, and resuspended inDMEM medium. For the cytotoxicity tests, MDPC cells were depositedin the lower chamber of the 24 well culture plate andleft for 4 hours at 37°C before any experiment.

Cytotoxicity tests

Cytotoxicity tests were performed with the Transwellinsert (Sigma-Aldrich, St. Louis, MO) methodologyand the immortalized mouse odontoblast cell line MD-

Table 1. Characteristics of tested materials.

Material Components LOT Manufacturer

Dycal Base paste: (1,3-butylene glycol disalicylate, zinc 120717 Dentsply Tulsa Dental,oxide, calcium phosphate, calcium tungstate, iron Johnson City, TN, USAoxide pigments). Catalyst paste: (calcium hydroxide, N-ethyl-o/p-toluenesulphonamide, zinc oxide, titanium oxide, zinc stearate,iron oxide pigments).

ProRoot MTA Powder: calcium phosphate, calcium oxide, silica, bismuth oxide. 12001879 Dentsply Tulsa Dental,Liquid: distilled water Johnson City, TN, USA

MTA-Angelus Powder: potassium oxide, aluminum oxide, sodium oxide, iron 24120 Angelus, Londrina, oxide, sulfur trioxide, calcium oxide, bismuth oxide, magnesium PR, Braziloxide, potassium sulfate, sodium sulfate, silica. Liquid: distilled water

Biodentine Powder: tricalcium silicate, dicalcium silicate, calcium carbonate, B06562 Septodont, Saint-calcium oxide, iron oxide, zirconium oxide. Maur-des-Fosses,Liquid: calcium chloride, hydro soluble polymer. France

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PC-23. The advantage of using a non direct contacttest for the evaluation of the dental material citotoxici-ty is related to the fact that cells and materials areusually separated (35).Cytotoxicity of the four pulp-capping materials was as-sessed with MDPC-23 cells grown in the lower cham-ber of a 24-mm diameter Transwell plate with a 0,3mm pore size polycarbonate membrane (Sigma) (35).In order to standardize the samples, for the variousanalyzes and evaluations, the materials were placedon sterile paper disks of 0.5 cm in diameter. All testmaterials were prepared and mixed under sterile hoodfollowing preparation methods recommended by themanufacturer. The excess material was removed us-ing sterile spatula. Dycal, ProRoot MTA, MTA-Angelusand Biodentine were mixed on special glass platesand later will be placed with sterile carrier on paperdisks. The Transwell membrane of the inner chamber,filled with the paper disks, was then placed into thelower chamber of the 24 well culture plate each con-taining at the bottom 5x104 cells and incubated at 37°C in 5% CO2 atmosphere for 24 h, 48 h and 72 h, re-spectively. In order to improve the search, the per-centage of vitality of the cells was evaluated in threetime intervals: 24, 48 and 72 hours. Some wells wereincubated with only tissue culture media (negativecontrol) whereas others with a 10% dilution of 30%H2O2 (positive control). The vitality was assessed byAlamar blue test. For a further control, the percentageof vitality of murine odontoblasts, at 72 hours, was al-so assessed with the MTT assay (bromide 3-(4,5-di-methylthiazol-2-yl)-2, 5-diphenyltetrazolium). The vi-tality test to Alamar blue reagent acts as an indicatorof cell health, determining the reducing power in orderto measure quantitatively the proliferative capacity;the reagent was added in a ratio of 1:10 to the cellculture and then the cells were kept in the incubatorfor 3-4 hours at 37º C. The degree of fluorescenceand the relative values of absorbance were then ac-quired by reading in a spectrophotometer at a wave-length of 595 nm. The MTT test is a standard colori-metric assay for measuring the activity of enzymesthat reduce the MTT to formazan (a salt blue) in themitochondria, giving the substance a blue/purple col-or. This reaction is assessed and measured by thespectrophotometric reading of the sample, at a wave-length of 570 nm. Five replicates for each pulp cup-ping material were used for each experiment per-formed in duplicate.

Confocal Laser Scanning Microscope (CLSM)

Once performed the cytotoxicity test of the differentmaterials, the Transwell inserts was removed and theland was eliminated from the culture plate. Afterwashing the slides with the buffer Buffer-TES, 250 mlof 10 mM solution of the fluorescent dye PSVueTM480 were added per well, in order to detect thepresence of apoptotic cells present in the culture.Apoptosis is defined as programmed, physiologicalcell death and plays an important role in tissue home-

ostasis. The loss of plasma membrane asymmetry isan early event in apoptosis, independent of cell type,resulting in the exposure of phosphatidylserine (PS)residues at the outer plasma membrane leaflet (36).PSVue reagents are a family of fluorescent probescontaining a bis(zinc2+dipicolylamine) group (Zn-DPA), a motif that has been found to bind with highaffinity to surfaces enriched with anionic phospho-lipids, especially phosphatidylserine (PS) exposed oncell membranes. The plate was kept under gentle agi-tation for 2 hours at room temperature. After 2 hours,the solution of PSVue has been eliminated and thewashing of the plate has been carried out with abun-dant Buffer-TES. The next step involved the additionof the dye Hoechst 33342, affine to DNA for viablecells. After 15 minutes the images were acquired us-ing confocal laser scanning microscope (CLSM) (37).

Results

Cytotoxicity tests

Figure 1 shows the results obtained with the Alamarblue test at 24, 48 and 72 hours. The results obtained to 24 hours show that the higherpercentage of cell vitality is found in Biodentine(106%), which shows an average of even greatercompared to the negative control cells, while Dycal(8.6%) is the material that presents the lowest values,so as to become the minimum value of the range ofvitality of the pulp-capping materials tested in re-search. ProRoot MTA and MTA-Angelus both showgood percentage of vitality, which amounted to 95%and 93.6% respectively. In the assessment performed at 48 hours, dissimilarresults emerge between the various materials. Someof them show an improvement of the percentage ofvitality; MTA-Angelus equals the number of cells ofthe negative control and Biodentine presents a cell vi-tality greater than 13% compared to control. Contrari-wise Dycal and ProRoot MTA show a deterioration ofthe data. In the assessment at 72 hours, the analysis of thesamples show a worst general behavior of the materi-als which leads to a decrease in the percentage of vi-tality and in the average number of cells. The only ex-ceptions are Biodentine and Dycal. Biodentine is thematerial with the best percentage compared to thenegative control, thus demonstrating a marked bio-compatibility. The average number of cells remainsstable compared to the previous assessment made at48 hours with a percentage which stabilizes at 114%.Dycal demonstrates a slight increase in the numberof cells (6%), with a substantially cytotoxic behavior.ProRoot MTA and MTA-Angelus prove to have aslight negative trend, but with good percentage of vi-tality that are stabilized on 71% for both materials.Figure 2 shows the results of the vitality tests per-formed with the MTT assay at 72 hours. The MTT testconfirmed the percentage ratios between the various

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after incubation with different pulp capping materialsconfirmed the cytotoxicity tests results: a few cellswere observed in the presence of Dycal, indicating anhigh level of citotoxicity (Fig. 5) whereas ProRootMTA (Fig. 6), MTA-Angelus (Fig. 7) and Biodentine(Fig. 8) did not seem to be cytotoxic.

Statistical analysis

As reported in Table 2, after 24 hours the amount ofcells present in contact with MTA-Angelus is not sta-

materials and between the materials and the posi-tive/negative controls determined with the Alamarblue test.. MTA Angelus shows the best percentageof vitality at all. In general even if the relationshipsbetween the various materials are similar, there wasa slight increase in the mean number of cells.

Confocal Laser Scanning Microscope (CLSM)

images

After staining with PSVueTM480 and Hoechst 33342dyes, the morphological structure of the cells in cul-ture was observed with CLSM. The use of fluorescentdye PSVueTM480 shows the presence of apoptoticcells. The Hoechst 33342 dye acts by binding to theDNA of viable cells and coloring the nucleus in blue.Figure 3 shows the negative control observations(preparation containing only the culture medium)while Figure 4 shows the positive control observa-tions (prepared with hydrogen peroxide added to theculture medium). As clearly shown, H2O2 is very cyto-toxic and the cells stained with PSVue480™ reagentare completely fluorescent in green (Fig. 3). In ab-sence of any type of materials, the cells were notgreen fluorescent (Fig. 4) but we could see the nucleistained with Hoechst. Figures 5 - 8 show the imagesacquired for each material. These images obtained

Figure 4. CLSM images of apoptosis assay in presence ofculture medium only (negative control).

Figure 3. CLSM images of apoptosis assay in the transwellwells prepared with a 30% solution of hydrogen peroxide(positive control).

Figure 1. Alamar blue test results at 24, 48 and 72 hours.

Figure 2. Results of the vitality test performed with the MTTassay at 72 hours.

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Figure 5. CLSM images of apoptosis assay in the transwellwells prepared with Dycal.

Figure 6. CLSM images of apoptosis assay in the transwellwells prepared with ProRoot MTA.

Figure 7. CLSM images of apoptosis assay in the transwellwells prepared with MTA-Angelus.

Figure 8. CLSM images of apoptosis assay in the transwellwells prepared with Biodentine.

Table 2. Mean ± standard deviation of Bonferroni post-hoc test of the different values of cell viability. Different superscriptletters indicate a statistically significant difference (P <0.001). The same superscript letter indicates a not statistically signifi-cant difference (P> 0.001).

Materials 24 h 48 h 72 h

Negative control 500000 ± 0a 522000 ± 0d 466000 ± 0g

Positive control (H2O2) 37000 ± 2738b 25000 ± 1850e 18000 ± 1332i

Dycal 43000 ± 2326c 22000 ± 5740e 30000 ± 1868l

ProRoot MTA 475000 ± 53675a 465000 ± 55629d 333000 ± 33157h

MTA-Angelus 468000 ± 72158a 522000 ± 56089d 333000 ± 59216h

Biodentine 533000 ± 60897a 592000 ± 20182d 533000 ± 42179f

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tistically different from the amount of cells present incontact with Biodentine, ProRoot MTA and the nega-tive control (P> 0001). The lowest values after 24 h isprovided by Dycal and the positive control. After 48 hours no statistically significant difference isdemonstrated between MTA-Angelus, Biodentine,ProRoot MTA and the negative control (P> 0.001). Nostatistically significant difference is also present be-tween Dycal and the positive control (P> 0.001),where the number of live cells is significantly lowercompared to the samples of the remaining materials(P <0.001). After 72 hours Biodentine shows a significantlygreater number of viable cells compared to all othermaterials tested (P <0.001), MTA-Angelus and Pro-Root MTA show values not significantly different be-tween them (P> 0.001) and the negative control hasretained intermediate values. Finally in the positivecontrol samples the lower number of cells was found(P <0.001).Table 3 shows the values obtained with the MTT test.There are no statistically significant differencesamong ProRoot MTA, MTA-Angelus, Biodentine andthe negative control (P>0.001). Dycal shows signifi-cant lower values (P<0.001).

Discussion and conclusion

Pulp capping materials should act as a barrier whichprotects the vitality of the entire pulp tissue by cover-ing the minimal exposed tissue and by preventingfrom further endodontic treatments. Due to this factthe material used should provide an appropriate hostresponse; this means that tissues that come into con-tact with the materials do not show any toxic, irritat-ing, inflammatory, allergic, genotoxic, or carcinogenicaction (38). In the present study Dycal demonstrates the lowerrates of vitality and a strong cytotoxic capability. Dycalhas shown the lowest mean number of cells in the col-orimetric assay performed with Alamar blue, with as-sessments at 24, 48 and 72 hours, and in the MTT as-say at 72 hours. The low percentage of vitality of Dy-cal occurs already in the first 24 hours, manifestingsmall variations of 1-2 percentage points to the vari-ous measurement intervals. These results confirm the

conclusions of others Authors (39, 40) on the non-complete biocompatibility of calcium hydroxide-basedmaterials: the protective effect of these materials to-wards the pulp is not complete. Calcium hydroxidehas an important action in protecting the pulp fromthermal, mechanical and microbiological stimuli (5, 6)because of its antibacterial action and its property ofstimulating sclerotic an reparative dentin formation. Inclinical practice, the presence of hard tissue barrier af-ter capping can be considered an asset, since it pro-vides natural protection against the infiltration of bac-teria and chemical products. However, the importanceof calcified hard tissue barrier formation after cappinghas been challenged by other studies, which haveshown multiple tunnel defects and cell inclusions inbridges following pulp capping with calcium hydroxide(41). This may lead to leakage and bacteria penetra-tion into pulp tissue unlike the permanent seal pro-duced by bonding agents. Furthermore it is equallydemonstrated that, due to the alkalinity of its pH, calci-um hydroxide induces cytotoxicity, causing the forma-tion of a layer of coagulation necrosis, when it is in di-rect contact with the dental pulp (7). For both thesereasons calcium hydroxide do not seem the eligiblematerial to be used in case of exposed pulp tissue. Very different results were obtained from the analysisof the MTA-based materials (ProRoot MTA and MTA-Angelus). Both materials to the evaluation of the 72hours, with Alamar blue test and MTT assay, have re-ported excellent percentage of vitality, detected in arange that goes from 71% to 95% of vitality and insome samples the results were even assimilated tothe negative control. This significant difference be-tween the values of vitality of calcium hydroxide andMTA is clearly due to the structural difference of thetwo basic components and due to the various physio-logical and biochemical reactions induced on tissues.It has been demonstrated that MTA has the ability toinduce the formation of a bridge of hard tissue ofgreater thickness compared to the bridge establishedin presence of calcium hydroxide, also managing tocause less inflammation in the adjacent tissues (18,20). The dental pulp also contains progenitor cellsand stem cells, which can proliferate and differentiateinto odontoblasts forming dentin; Guven and Cehreli(42) reported that, probably, MTA is able to facilitatethese cellular changes by inducing the secretion ofmorphogenetic proteins and growth factors such asBMP-2 and TGF-β1.In the present study Biodentine proved to be themore biocompatible material. Biodentine, in measure-ments made at 24, 48 and 72 hours, reported per-centage of vitality above the negative control. After24 hours it recorded values equal to 106%, rose to113% and 114% in the two subsequent analysis. Bio-dentine is a new bioactive cement based on calciumsilicate for pulp capping, derivation of bioengineering,with anti-inflammatory behavior (43), different fromthe classic materials based on calcium silicate, suchas Portland cement. The technology behind the man-ufacturing process of the active bio-silicate, the mainconstituent of Biodentine, removes the metallic impu-

Table 3. Mean ± standard deviation of Bonferroni post-hoctest of the different values of cell viability. Different super-script letters indicate a statistically significant difference (P<0.001). The same superscript letter indicates a not statis-tically significant difference (P> 0.001).

Materials MTT

Negative control 124074 ± 0a

Dycal 45741 ± 5040b

ProRoot MTA 140741 ± 30098a

MTA-Angelus 179444 ± 99142a

Biodentine 166481 ± 59317a

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rities which are present in other cements (44). Thesetting reaction involves the hydration of tricalciumsilicate, the production of a calcium silicate-based geland calcium hydroxide, which in contact with phos-phate ions, it is able to create precipitated similar tohydroxyapatite.Considering the interface between dentin and Bioden-tine with confocal microscopy, Atmeh et al. (45) showedthat microstructural changes occur in this area with anincreased content of carbonate at the dentin interface.These observations suggest how the intertubularspread produced by Biodentine hydration lead to thecreation of a hybrid layer (46). Furthermore, histologicalevaluations carried out on samples prepared with Bio-dentine have demonstrated the ability of the material toinduce the differentiation of odontoblasts starting frompulp progenitor cells, forming a mineralizing matrix withthe characteristics of dentin (47). In conclusion Biodentine has shown to be the materi-al with the best qualities and characteristics, that arethe basis of biocompatibility. Because of the lower cy-totoxicity and the higher bio-inductive ability, Bioden-tine can be considered an ideal cement for pulp-cap-ping. Nevertheless, especially for the new generationmaterials, further studies must be started to demon-strate the clinical efficacy and illustrate the actualmechanisms of action, both in vitro and in vivo.

Acknowledgments

Nothing to declare.

Conflict of interest statement

The authors of this study have no conflict of interestto disclose.

References

1. European Society of Endodontology. Quality guidelines forendodontic treatment: consensus report of the European So-ciety of Endodontology. International Endodontic Journal.2006;39:921-30.

2. Obeid M, Saber Sel D, Ismael Ael D, Hassanien E. Mes-enchymal stem cells promote hard-tissue repair after directpulp capping. Journal of Endodontics. 2013;39(5):626-31.

3. Dominguez MS, Witherspoon DE, Gutmann JL, OppermanLA. Histological and scanning electron microscopy assess-ment of various vital pulp-therapy materials. Journal of En-dododontics. 2003;29:324-33.

4. Camilleri J, Pitt-Ford TR. Mineral trioxide aggregate: a re-view of the constituents and biological properties of the ma-terial. International Endodontic Journal. 2006;39:747-54.

5. Desai S, Chandler N. Calcium hydroxide-based root canalsealers: a review. Journal of Endodontics 2009; 39:415-22.

6. Mohammadi Z, Dummer PMH. Properties and applicationsof calcium hydroxide in endodontics and dental traumatol-ogy. International Endodontic Journal. 2011;40:697-730.

7. Duda S, Dammaschke T. Maßnahmen zur Vitalerhaltung derPulpa. Gibt es Alternativen zumKalziumhydroxid bei der di-rekten Überkappung? Quintessenz. 2008;59:1327-34.

8. Duda S, Dammaschke T. Die direkte Überkappung – Vo-raussetzungen für klinische Behandlungserfolge. Endodontie.2009;18:21-31.

9. Hørsted-Bindslev P, Vilkinis V, Sidlauskas A. Direct pulp cap-ping of human pulps with a dentin bonding system or withcalcium hydroxide cement. Oral Surgery Oral Medicine OralPathology Oral Radiology & Endodontology. 2009;96:591-600.

10. Goldberg M, Six N, Decup F, Lasfargues JJ, Salih E, Tomp-kins K, Veis A. Bioactive molecules and the future of pulptherapy. American Journal of Dentistry. 2003;16:66-76.

11. Almushayt A, Narayanan K, Zaki AE, George A. Dentin ma-trix protein 1 induces cytodifferentiation of dental pulp stemcells into odontoblasts. Gene Therapy. 2006;13:611-20.

12. Furey A, Hjelmhaug J, Lobner D. Toxicity of Flow Line, Du-rafill VS, and Dycal to dental pulp cells: effects of growth fac-tors. Journal of Endodontics. 2011;36:1149-53.

13. Shen Q, Sun J, Wu J, Liu C, Chen F. An in vitro investiga-tion of the mechanical-chemical and biological properties ofcalcium phosphate/calcium silicate/bismutite cement for den-tal pulp capping. Journal of Biomedical Materials ResearchPart B: Applied Biomaterials. 2010;94:141-8.

14. Torabinejad M, Parirokh M. Mineral trioxide aggregate: a com-prehensive literature review part ii: leakage and biocom-patibility investigations. Journal of Endodontics. 2013;36:190-202.

15. Takita T, Hayashi M, Takeichi O, Ogiso B, Suzuki N, Otsu-ka K, Ito K. Effect of mineral trioxide aggregate on prolifer-ation of cultured human dental pulp cells. International En-dodontic Journal. 2006;39:415-22.

16. Sawicki L, Pameijer CH, Emerich K, Adamowicz-Klepalska B.Histological evaluation of mineral trioxide aggregate and cal-cium hydroxide in direct pulp capping of human immature per-manent teeth. American Journal of Dentistry. 2008;21:262-6.

17. Gandolfi MG, Shah SN, Feng R, Prati C, Akintoye SO. Bio-mimetic calcium-silicate cements support differentiation ofhuman orofacial bone marrow stromal cells. Journal of En-dodontics. 2011;37:1102-8.

18. Moghaddame-Jafari S, Mantellini MG, Botero TM, McDon-ald NJ, No¨r JE. Effect of ProRoot MTA on pulp cell apop-tosis and proliferation in vitro. Journal of Endodontics.2005;31:387-91.

19. Okiji T, Yoshiba K. Reparative dentinogenesis induced bymineral trioxide aggregate: a review from the biological andphysicochemical points of view. International Journal of Den-tistry. 2009;34:1-12.

20. Hauman CHJ, Love RM. Biocompatibility of dental materi-als used in contemporary endodontic therapy: a review. Part2. Root canal-filling materials. International EndodonticJournal. 2003;36:147-60.

21. Asgary S, Shahabi S, Jafarzadeh T, Amini S, Kheirieh S. Theproperties of a new endodontic material. Journal of En-dodontics. 2008;34:990-3.

22. Gandolfi MG, Pagani S, Perut F, Ciapetti G, Baldini N, Mon-giorgi R, Prati C. Innovative silicate-based cements for en-dodontics: a study of osteoblastlike cell response. Journalof Biomedical Materials Research. 2008;87:477-86.

23. Gomes-Filho JE, Rodrigues G, Watanabe S, Estrada Bern-abé PF, Lodi CS, Gomes AC, Faria MD, Domingos Dos San-tos A, Silos Moraes JC. . Evaluation of the tissue reactionto fast endodontic cement (CER) and Angelus MTA. Jour-nal of Endodontics. 2009;35:377-80.

24. Johnson BR. Considerations in the selection of a rootend fill-ing material. Oral Surgery Oral Medicine Oral Pathology OralRadiology & Endodontology. 1999;87:398-404.

25. Camilleri J. Characterization of hydration products of min-eral trioxide aggregate. International Endodontic Journal.2008;41:408-17.

© C

IC Ed

izion

i Int

erna

ziona

li

Annali di Stomatologia 2014; V (2): 69-7676

C. Poggio et al.

26. Camilleri J. Characterization and hydration kinetics of tri-calcium silicate cement for use as a dental biomaterial. Den-tal Materials. 2011;27:836-44.

27. Tay FR, Pashley DH, Rueggeberg FA, Loushine RJ, WellerRN. Calcium phosphate phase transformation produced bythe interaction of the Portland cement component of whiteMineral Trioxide Aggregate with a phosphate-containing flu-id. Journal of Endodontics. 2007;33:1347-51.

28. Taddei P, Inti A, Gandolfi MG, Possi PML, Prati C. Ageingof calcium silicate cements for endodontic use in simulatedbody fluids: a micro-Raman study. Journal of Raman Spec-troscopy. 2009;40:1858-66.

29. Han L, Okiji T, Okawa S. Morphological and chemical analy-sis of different precipitates on mineral trioxide aggregate im-mersed in different fluids. Dental Materials Journal.2010;29:512-7.

30. Schmalz G. Use of cell cultures for toxicity testing of dentalmaterials – advantages and limitations. Journal of Dentistry.1994;22(Suppl. 2):6-11.

31. Parirokh M, Torabinejad M. Mineral trioxide aggregate: acomprehensive literature review-Part I: chemical, physical,and antibacterial properties. Journal of Endodontics. 2010;36:16-27.

32. Parirokh M, Torabinejad M. Mineral trioxide aggregate: a com-prehensive literature review-Part III: clinical applications, draw-backs, and mechanism of action. Journal of Endodontics.2010;36:400-13.

33. Mac Dougall M, Selden JK, Nydegger JR and Carnes DL.Immortalized mouse odontoblast cell line M06-G3 applica-tion for in vitro biocompatibility testing. American Journal ofDentistry. 1998;10:11-6.

34. de Souza Costa CA, Vaerten MA, Edwards CA and HanksCT. Cytotoxic effects of current dental adhesive systems onimmortalized odontoblast cell line MDPC-23. Dental Mate-rials. 1999;15:434-41.

35. Babich H, Sinensky MC. Indirect cytotoxicity of dental ma-terials: a study with Transwell inserts and the neutral red up-take assay. Alternative to Laboratory Animals. 2001;29(1):9-13.

36. van Engeland M, Nieland LJW, Ramaekers FCS, Schutte B,Reutelingsperger CPM. Annexin v-affinity assay: a review

on an apoptosis detection system based on phos-phatidylserine exposure. Cytometry. 1998;31:1-6.

37. Masuda-Murakami Y, Kobayashi M, Wang X, Yamada Y,Kimura Y, Hossain M, Matsumoto K. Effects of mineral tri-oxide aggregate on the differentiation of rat dental pulp cells.Acta Histochemistry. 2010;112(5):452-8.

38. Valey JW, Simonian PT, Conrad EU. Carcinogenicity andmetallic implants. American Journal of Orthododontics andDental Orthopedics. 1995;24:319-24.

39. Aeinehchi M, Eslami B, Ghanbariha M, Saffar AS. Mineraltrioxide aggregate (MTA) and calcium hydroxide as pulp-cap-ping agents in human teeth: a preliminary report. InternationalEndodontic Journal. 2002;36:225-231.

40. Briso ALF, Rahal V, Mestrener SR, Dezan E Jr. Biologicalresponse of pulps submitted to different capping materials.Brazilian Oral Research. 2006;20:219-225.

41. Goldberg F, Massone EJ, Spielberg C. Evaluation of the denti-nal bridge after pulpotomy and calcium hydroxide dressing.Journal of Endododontics. 1984;10(7):318-20.

42. Guven G, Cehreli ZC, Ural A, Serdar MA, Basak F. Effectof mineral trioxide aggregate cements on transforminggrowth factor b1 and bone morphogenetic protein produc-tion by human fibroblasts in vitro. Journal of Endododontics.2007;33:447-450.

43. About I, Laurent P, Tecles O. Bioactivity of biodentine: aCa3SiO5-based dentin substitute. Journal of Dental Research.2010;89:165.

44. Grech L, Mallia B, Camilleri J. Characterization of set In-termediate Restorative Material, Biodentine, Bioaggregateand a prototype calcium silicate cement for use as root-endfilling materials. International Endodontic Journal.2013;46:632-41.

45. Atmeh AR, Chong EZ, Richard G, Festy F, Watson TF.Dentin-cement interfacial interaction: calcium silicates andpolyalkenoates. Journal of Dental Research. 2012; 91:454-9.

46. Colon P, Bronnec F, Grosgogeat B, Pradelle-Plasse N. In-teraction between a calcium silicate cement (Biodentine) andits environment. Journal of Dental Research. 2010;89:401.

47. About I. Dentin regeneration in vitro: the pivotal role of sup-portive cells. Advanced Dental Research. 2011;23:320-324.

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